Typhoid fever is a major cause of morbidity worldwide with an estimated incidence of 16 to 33 million infections and 500,000 to 600,000 deaths annually. It is caused by Salmonella typhi (S. typhi). The Vi capsular polysaccharide of S. typhi is the protective antigen and is used in current licensed Vi vaccines for prophylactic immunization against typhoid fever for people of e 2 years. The current licensed Vi vaccines are produced by costly and hazardous fermentation of S. typhi wild type bacteria and elaborate purification processes. A second generation of typhoid vaccines based on Vi polysaccharide-protein conjugate is being developed that may potentially be more immunogenic and effective in children under 2 years. A high-molecular-weight PGA (HPGA) from a plant source has been developed and is manufactured by Nanotherapeutics under cGMP at a kilogram (kg) scale. The end product also had a high molecular weight (>2 x 106 Da) similar to that of the native Vi polysaccharide. The O-acetylated HPGA (OAcHPGA) not only shared the same antigenicity with the Vi polysaccharide, but was also immunogenic in laboratory animals. The antibody response level induced by OAcHPGA was comparable to that of a marketed Vi vaccine. These studies indicate that this OAcHPGA could potentially be used to make a synthetic typhoid vaccine that can be produced in large quantities from a plant-based starting material (HPGA). Compared to the existing Vi polysaccharide vaccine, OAcHPGA could be a much safer and less expensive vaccine. The economic advantage makes it easier and more affordable to expand production and use of the typhoid vaccine worldwide, especially in endemic areas of developing countries. Thus, we propose to continue developing a synthetic typhoid vaccine based on OAcHPGA with the following two specific aims: (Specific Aim 1 - Immunogenicity and protection studies) To evaluate the immunogenicity of OAcHPGA based on the potency indicators (DOAc and molecular weight) and to demonstrate the protective effect of the OAcHPGA against the S. typhi challenge in animal models. This will establish that the OAcHPGA is immunogenic and protective against the S. typhi, and help establish product specifications in the future. (Specific Aim 2 - Process development) To refine and establish the O-acetylation process for producing OAcHPGA with desired DOAc, molecular weight, and other properties. This will be conducted together with related assay development and in conjunction with Specific Aim 1. In addition, the OAcHPGA-protein conjugate will be prepared and evaluated. Accomplishment of these two specific aims will form the base for next development phases toward pilot cGMP manufacturing, animal toxicology studies, and clinical studies of the OAcHPGA vaccine and, also importantly, the development of an OAcHPGA-protein conjugate vaccine that may potentially be more immunogenic and suitable for children under the age of 2. PUBLIC HEALTH RELEVANCE: Typhoid fever is a major cause of morbidity worldwide with an estimated incidence of 16 to 33 million infections and 500,000 to 600,000 deaths annually. The current licensed Vi vaccines are produced by costly and hazardous fermentation of S. typhi wild type bacteria and elaborate purification processes. A second generation of typhoid vaccines based on Vi polysaccharide-protein conjugate is being developed that may potentially be more immunogenic and effective in children under 2 years.